The reemergence of smallpox (or derivative) in an unprotected civilian or military population would likely cause mass casualties, panic, and functional breakdown. Thus, the availability of a safe effective poxvirus vaccine may significantly alleviate some of these concerns. Among the poxvirus-encoded proteins, four membrane proteins (A33R, B5R, L1R and A27L) (called 4-pox vaccine) have been shown to generate protective neutralizing antibody responses and host range protein 2 (HRP2, also called 018L) has been shown to generate protective T cell-mediated immune response against poxvirus challenge in HLA-A2 transgenic mice. DNA vaccines targeting these key smallpox antigens represent a potentially plausible approach for the control of smallpox infections. A limitation of this DNA vaccination strategy is the naturally low immunogenicity of DNA encoding antigen, resulting in limited vaccine potency. Intradermal administration of DNA vaccines via gene gun represents an efficient method for delivery of DNA vaccines into professional APCs in vivo. The focus of the present application is to investigate different methods of enhancing potency based upon strategies that target the APCs. By selecting five methods of immune enhancement that rely on different mechanisms of improving antigen presentation, it is hoped that novel additive or synergistic effects will be elicited to generate effective immunity. These methods include: 1) codon optimization of the 4-pox antigens to enhance antigen expression;2) linkage of poxvirus antigen to calreticulin (CRT) to enhance antigen processing and presentation;3) co-administration with DNA encoding heat labile enterotoxin (LT) from enterotoxigenic E. coli to enhance immunogenicity;4) co-administration with a plasmid expressing GM-CSF to attract APCs and adjuvant DNA vaccines;and 5) addition of a DNA vaccine encoding a documented target protein of cell-mediated immunity (i.e. poxvirus HRP2 protein). Thus, in the current application, we will test if intradermal delivery of a codon-optimized 4-pox DNA vaccines and/or HRP2 DNA adjuvanted by inclusion of GM-CSF or LT DNA and/or linkage to CRT via gene gun would lead to enhanced antigen-specific humoral and T cell-mediated immune responses and antiviral effects against live poxvirus infections in vivo. The successful implementation of the proposed studies will result in an improved poxvirus DNA vaccine potentially suitable for clinical development.